基于“脾气散精”理论研究健脾化湿法调控肝脏脂质输出基因甲基化治疗NAFLD机制

NAFLD is the most common risk factor of diabetes mellitus and cardiovascular disease.With the improvement of living level,the prevalence of NAFLD is increased rapidly. Studies have shown that DNA methylation is closely related to NAFLD. MTP is a key enzyme for transporting liver lipids outward. The increase of MTP promoter methylation level is an important factor for liver lipid deposition of NAFLD. Chinese medicine believes that the "spleen Qi transfers essence ", MTP lipid transport function may be one of the manifestations for "spleen qi" .Clinical research shows that “ nourishing spleen and dispelling dampness” is an important principle in the treatment of NAFLD, but the mechanism has not been elucidated. In this study, Shenlingbaizhu powder is used as the carrier of the principle for nourishing spleen and dispelling dampness,and in vivo and in vitro experiments of NAFLD are used to investigate whether the principle for nourishing spleen and dispelling dampness can regulate the level of hepatic total methylation and the MTP promoter methylation to treat NAFLD. The results will explain the scientific connotation of "spleen qi transfers essence " and the mechanism of the principle for nourishing spleen and dispelling dampness to treat NAFLD.

NAFLD是糖尿病、心脑血管疾病最常见危险因素，随着生活水平提高，NAFLD患病率急剧上升。研究表明，DNA甲基化与NAFLD关系密切。MTP是肝脏脂质向外输出的关键酶，MTP启动子甲基化水平增加，是NAFLD肝脏脂质沉积重要因素。中医认为“脾气散精”， MTP脂质输出功能可能是“脾气散精”的表现形式之一，临床研究表明，健脾化湿法是治疗NAFLD的重要原则，但其机制尚未阐明。本课题以参苓白术散作为健脾化湿法的载体，采用NAFLD在体和离体模型，利用实时定量PCR和免疫组化等方法检测总甲基化、DNMT、MTP的表达及MTP甲基化水平，研究健脾化湿法能否通过调控肝脏总甲基化水平和MTP启动子甲基化水平达到治疗NAFLD的目的。研究结果将阐释“脾气散精”的科学内涵及健脾化湿法治疗NAFLD的机制。

NAFLD严重危害人类健康。研究表明MTP启动子甲基化修饰增加，是NAFLD肝脏脂质沉积重要因素。中医认为“脾气散精”，甘油三酯作为精微的一种表达形式，MTP脂质输出功能是“脾气散精”的表现形式之一。本课题拟以参苓白术散作为健脾化湿法的载体，通过 NAFLD 体内外模型，研究健脾化湿法能否通过调控肝脏总甲基化水平和MTP启动子甲基化水平达到治疗NAFLD的目的，阐释“脾气散精”的科学内涵及健脾化湿法治疗NAFLD的机制。研究结果显示：1、参苓白术散明显改善NAFLD大鼠的脂质积累，随着参苓白术散剂量的增加，肝脏脂滴生成减少，组织结构明显改善。肝脏及血清脂质和转氨酶水平明显下降。2、参苓白术散可以上调NAFLD大鼠引起的MTP、APOB、LDLR mRNA 的表达下降；下调NAFLD大鼠甲基转移酶Dnmt1、DNMT3b、Dnmt3a以及MeCP2mRNA的表达升高；下调NAFLD大鼠肝脏Mecp2的阳性表达上升，同时上调MTP阳性细胞表达，显著降低MTP的甲基化程度。3、参苓白术散可以改善PA诱导HepG2细胞的脂质沉积，下调DNMT1、DNMT3a、DNMT3b、MeCP2mRNA表达升高；上调MTP、ApoB、LDLRmRNA表达降低, 从而改善HepG2细胞的脂质积累，促进脂质代谢。4、代谢组学结果显示，参苓白术散通过调控缬氨酸、亮氨酸和异亮氨酸生物合成，缬氨酸、亮氨酸和异亮氨酸降解，氨酰-tRNA生物合成等物质代谢，减轻肝脏脂质沉积治疗NAFLD。5、蛋白质组学结果显示，参苓白术散通过调控代谢通路、PPAR信号通路以及视黄醇代谢等通路，调节NAFLD的脂质代谢。综上健脾化湿法通过调控肝脏总甲基化水平和MTP启动子甲基化水平治疗NAFLD，阐释了“脾气散精”的科学内涵及治疗NAFLD的机制。